DTIC ADA368460: Action of the p53 Effector, p2l, on its Targets: Cyclin cdk and PCNA

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AWARD NUMBER DAMD17 -97-1-73 14

TITLE: Action of the p53 Effector, p21, on its targets: Cyclin-
cdk and PCNA

PRINCIPAL INVESTIGATOR: Anindya Dutta, M.D.,Ph.D.

CONTRACTING ORGANIZATION: Brigham and Women's Hospital
, Boston, Massachusetts 02115

REPORT DATE: October 1998

TYPE OF REPORT: Annual

PREPARED FOR: U.S. Army Medical Research and Materiel Command
Fort Detrick, Maryland 21702-5012

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Annual (22 Sep 97 - 21 Sep 98)

4. TITLE AND SUBTITLE

Action of the p53 Effector, p21, on It's Targets: Cyclin-cdk and PCNA

5. FUNDING NUMBERS
DAMD17-97-1-7314

6. AUTHOR(S)

Anindya Dutta, M.D.,Ph.D.

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)
Brigham and Women's Hospital
Boston, Massachusetts 02115

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U.S. Army Medical Research and Materiel Command
Fort Detrick, Maryland 21702-5012

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AGENCY REPORT NUMBER

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13. ABSTRACT (Maximum 200 words!

CXm aim is to dissect the interactions of p21 (a downstream effector of the tumor suppressor protein
p53) Y^th its two target, cyclin-cdks and PCNA. In year 1 we have estabUshed conditions for
quantitatively analyzing the inhibition of cyclin-cdk by p21. This wiU enable us to measure the
exact contribution of the interaction between the cyclin-binding Cy motif of p21 with a docking site
on the cyclin in the inhibition of kinase activity. The conditions obtained have already
demonshated qualitatively the importance of the Cy motif in kinase inhibition. Different methods
of site directed mutogenesis are teing tried to determine the optimal method by which to generate a
hbrary of mutants in the Cy motif of p21. In year 2 this library of mutants will enable us to
quantitatively deteimine exactly what constitutes a functional Cy motif. In order to dissect the
mteraction of p21 with PCNA we now have mutant forms of PCNA that fail to interact with p21 or
with the exonuclease Fenl without disrupting the trimeric structure of PCNA. This is the first step
towards analyzing how the p21-PCNA interaction impinges on DNA replication and repair in
cancer cells.

14. SUBJECT TERMS
Breast Cancer PCNA
P21 Cell Cycle

p53

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adhered to the "Guide for the Care and Use of Laboratory
Animals, " prepared by the Committee on Care and use of Laboratory
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adhered to policies of applicable Federal Law 45 CFR 46.

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investigator (s) adhered to current guidelines promulgated by
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conduct of research utilizing recombinant DNA, the
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Involving Recombinant DNA Molecules.

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TABLE OF CONTENTS PAGE

INTRODUCTION 2

BODY 3

METHODS 4

RESULTS AND DISCUSSION 5

CONCLUSION 7

FIGURE LEGENDS 7

FIGURES 8

REFERENCES 13

1

INTRODUCTION:

Importance of p53 function in breast cancer: As discussed extensively in the
original grant proposal, the p53 protein is an important tumor suppressor which is
inactivated by mutation in up to 50% of breast cancers. In multiple studies, mutation of the
p53 gene in a breast cancer is associated with shorter disease free interval and decreased
overall survival, independent of the presence of axillary node metastases. In a small group
of familial breast cancer patients (with Li Fraumeni syndrome), inactivating germ-line
mutations are seen in the p53 gene, indicating the importance of normal p53 in preventing
the appearance and progression of breast cancers.

p53 and n21: Both the germinal and the somatic mutations of p53 which are seen
in cancers appear to be "loss of function" mutations. p53 is believed to suppress cell
growth by the transcriptional induction of genes that negatively regulate cell growth. One
such gene, discovered is the p21 gene (1).

p21 belongs to one class of cdk inhibitors which are related to each other in their
primary sequence and their substrate specificity. These associate with and inhibit almost all
cyclin-cdk pairs, and the three members of this class are p21, p27 and p57.

Domain structure of n21 : Beside inhibiting cyclin-cdk kinases, the p21 protein directly
interacts with and inhibits an essential DNA replication factor, proliferating cell nuclear
antigen (PCNA) (2-4). The N terminal domain of p21 (p21N) interacts with the cd^
protein and inhibits cyclin-cdk kinase activity, while the C terminal domain (p21C) interacts
with and inhibits PCNA, the ring shaped sliding clamp that tethers DNA polymerases delta
and epsilon to the replicating strand.

The cvclin bindinn motif in n21: Further dissection of the cdk inhibitory domain present in
p21N demonstrated that a cyclin binding motif on p21 allows it to directly bind to cyclins
(5). The cyclin binding sequence, ACRRLFGPV, is highly conserved in the other cdk
inhibitors, p27 and p57, and is also conserved in substrates and activators of cdks. A
twelve amino acid peptide containing this motif is sufficient to interact with cyclins.
Together with our biochemical data, the crystallographic structure of cyclin A-cdk2
complexed with p27 (6), suggests that the Cy motif- cyclin interaction serves as a docking
interaction essential for the complete interaction between cdk inhibitors and cyclin-cdk. In
this project we shall determine how the cyclin binding site of p21 contributes to inhibition
of cyclin-cdk kinase, and the implications of this discovery for designing chemicals that
mimic p21 action by binding cyclin or cdk2.

The, effect of p21 on the function of PCNA. PCNA inhibition by p21 is also important for
growth control (7, 8). We have recently shown that in serum-starved diploid fibroblasts,
the introduction of the PCNA inhibitory portion of p21 (without the cdk kinase inhibitor
domain) is also sufficient to inhibit cell growth (8). Hence, in the second portion of this
proposal we shall study the mechanism by which p21 affects the function of PCNA.

p21 disrupts the association of PCNA with the 5’-3’ exonuclease Fenl. Prokaryotic DNA
replication polymerases possess three activities: a 5’-3’ DNA polymerase, a 3’-5’
exonuclease (the proof-reading activity) and a 5’-3’ exonuclease. None of the DNA
polymerases identified in human cells possess the last activity. A 5’-3’ exonuclease is
however essential for complete DNA replication, and has been purified by virtue of its
requirement in the SV40 based DNA replication reaction (9). In the absence of this special
exonuclease the ribonucleotide-deoxyribonucleotide bond at the 5’ ends of Okazaki
fragments is not removed and consequently the Okazaki fragments are not ligated. This
exonuclease has been independently identified as a flap endonuclease (Fenl) required for
DNA recombination (10, 11) and as a gene essential for (a) cell-cycle progression and (b)

2

protection from radiation induced DNA damage in yeast S. cerevisiae (YKL510 or RAD27
or ERCl) and S. pombe (rad2) (12-14). Mutation of this gene in S. cerevisiae also
produces an instability of direct repeats much like the instability seen in several human

cancers (15). ,

We reported that Fenl/Rad2 directly associates with PCNA and that p21 disrupts
the association of Fenl with PCNA (16). This newly discovered activity of p21 is likely to
be important for the propagation of DNA mutations following DNA replication, because
unlinking Fenl from PCNA by p21 may promote the same type of genomic instability as
seen when RAD27 is mutated in S. cerevisiae. In this proposal we shall seek derivatives of
PCNA which have uncoupled the PCNA-pol delta and PCNA-Fenl interactions. These
studies will in addition illuminate whether the complete action of p21 on the DNA
replication apparatus requires that both these interactions of PCNA be breached. The
results will determine whether uncoupling of the polymerase from the exonuclease by p21
contributes to the genomic instability seen in several breast cancers and also indicate
whether the PCNA-Fenl complex could on its own be a suitable target for chemotherapy.

BODY

Original Statement of work

Technical objective 1. Analysis of the interaction between p21, cyclin and cdk.

Task 1: Months 1-12: Determination of the binding affinity of the cyclin-binding site for cyclins
and of the cdk2 binding site for cdk2 and comparison with the binding affinity of intact p21
with the cyclin E-cdk2 holoenzyme.

Task 2: Months 12-24: T.ineweaver-Burke analysis of the inhibition of cvclin E-cdk2 bv intact
p21 and bv p21 without the cvclin binding site.

Task 2: Months 24-36: Olipomicleotide directed mutagenesis of the portion of p21 in the
cyclin binding site to determine what sequence feature is essential for binding to cvclins.

Technical objective 2. Analysisoftheinteractionbetweenp21, PCNA and Fenl.

Task 5: Months 1-12: Determination of which part of PCNA interacts with n21 and with
Fenl.

The underlined tasks are being done now. We have slightly re-ordered our priorities in
Objective 1 because obtaining the mutant forms of the cyclin-binding motif (originally planned
for months 24-36) will significantly improve our measurements of binding affinity of cyclin-
binding sites for cyclins and the contribution of these sites to the association of p21 with
cyclin-cdks (Task 1) . Hence from this objective. Task 2 is being attempted now as Task 1 and
the original Task 1 has been moved back. This is reflected in the revised Statement of Work
below:

Technical objective 1. Analysis of the interaction between p21, cyclin and cdk.

Task 1: Months 1-24: Oligonucleotide directed mutagenesis of Ae portion of p21 in the cyclin
binding site to determine what sequence feature is essential for binding to cyclins.

Task 1: Months 1-24: Lineweaver-Burke analysis of the inhibition of cyclin E-cdk2 by intact
p21 and by p21 without the cyclin binding site.

3

Task 2: Months 24-36: Determination of the binding affinity of the cyclin-binding site for
cyclins and of the cdk2 binding site for cdk2 and comparison with the binding affinity of intact
p21 with the cyclin E-cdk2 holoenzyme.

Task 3: Months 24-48: Creation and testing of mutations of p21 with different affinities for
cyclins D1 and E.

Task 4: Months 24-48: Creation and testing of versions of p21 with variation of the distance
between the cyclin-binding and the cdk2 binding sites (in cis).

Technical objective 2. Analysis of the interaction between p21, PCNA and Fen 1.

Task 5: Months 1-12: Determination of which part of PCNA interacts with p21 and with
Fenl.

Task 6: Months 12-24: Creation of p21 derivatives and determination of their IC90 on the
processivity of PCNA-polymerase delta.

Task 7: Months 24-36: Creation and testing of PCNA mutants that have lost the interaction
with Fenl but can still stimulate polymerase delta

Task 8: Months 36-48: Testing effect of adding Fenl to replication reactions or over¬
expressing Fenl in MCF-7 cells in culture.

METHODS.

Inhibition of cyclin E-cdk2 by intact p21 and by p21 without the cyclin-
binding site.

The substrate used was GST-CDC25A produced in bacteria and purified on glutathione
agarose beads.. The enzyme (cyclin E-cdk2) was prepared by co-inf(^ting SF9 insect celk
with two baculoviruses expressing GST-cyclin E and cdk2. Active kinase complex is purified
on glutathione agarose beads. Both substrate and enzyme were eluted with glutathione.

To determine initial velocity conditions for the kinase reaction, 4 ng GST-cyclin E/cdk2
was incubated with 2 or 0.2 |Xg of GST-CDC25A in 25 |xl reaction buffer containing 50 mM

Tris-HCl pH 7.4, 10 mM MgC12, 1 mM DTT, 50 pM ATP containing 5 pCi ganama 32P-ATP
(sp. activity 3200 cpm/pmol). Reaction was allowed to proceed for indicated periods of time at
37 deg. c and stopped by boiling in Laemmli sample buffer. Products were separated by gel
electrophoresis, GST-CDC25A visualized by autoradiography, and radioactivity incorporated
into the band counted by liquid scintillation counting.

To determine conditions for inhibition by p21, bacterially produced GST-p21 was
purified by glutathione agarose affinity chromatography and titrated into the reaction (from 0.1

to 1000 ng). In addition p21 derivatives with deletions in the Cy motif (A17-24) and K motif

(A53-58) and 12 mer peptides containing the Cy motif (PS 100) and with a mutation in the Cy
motif (PS 101) were tested for their inhibition.

Mutagenesis of the Cy motif of p21.

The following strategies were tried in parallel.

4

1) A PCR-based strategy. Diverging oligonucleotides were made that annealed to the Cy motif
region of p21 but incorporated specific mutations. PCR with these oligonucleotides and others
that anneal to the ends of p21N cDNA generated fragments of 90 and 200 nucleotides which
were then annealed to each other (through their overlap at the Cy motif region and re-PCR-ed
with the end-specific oligonucleotides). The goal was to produce a 270 base-pair long
fragment that would contain the mutant Cy motif which would then be cloned into the pGEX
vector to produce GST-p21N with point-mutation in the Cy motif.

2) A cassette based strategy. PCR-based mutagenesis of GEX-p21N introduced a Hindlll site
at the N terminal end of the Cy motif (a silent mutation). Together with a naturally occurring
BlpI site on the C terminal side of the Cy motif, this allowed us to excise the wild-type Cy
motif. Synthetic oligonucleotides were annealed so that a mutant Cy motif was encoded by the
cassette and cloned between the Hindlll-BlpI sites of GEX-p21N.

3) The Kunkel method of mutagenesis. p21N was cloned into a vector (Blue-Script) that can
produce single-stranded DNA. deoxy-Uridine containing single-stranded DNA template was
prepared in CJ236 (dut, ung) bacteria. Annealing of mutant oligonucleotides (containing a
point-mutation in the Cy motif) to this template followed by E. coli Klenow polymerase
directed in vitro synthesis and transformation into wild-type (dut+„ ung+) bacteria is expected
to select against the deoxy-Uridine containing single-stranded DNA template so that most of the
colonies obtained will encode the mutant Cy motif.

Determination of the part of PCNA that interacts with p21 and Fenl.

A report appeared in the Literature (17) that indicated residues QI^I in the inter-domain
connecting loop of PCNA was important for die association of PCNA with p21 and
dispensable for forming a PCNA trimer. Plasmids encoding the mutant forms of PCNA
described in that report were obtained from the authors. Bacterially produced GST-p21 and to
GST-FenlC (containing the C terminal portion of Fenl which interacts with PCNA) were
bound to glutathione agarose beads and their association with PCNA assayed as described by
us (16). The wild type and mutant forms of PCNA were produced in two ways: (a) by in
vitro-transcription translation in rabbit reticulocyte lysates and (ii) by expressing in E. coli.
PCNA produced by in vitro transcription-translation was labeled with 35S methionine and
visualized by fluorography, while the bacterially produced PCNA was visualized by
immunoblotting with commercial anti-PCNA antibody.

RESULTS AND DISCUSSION

Inhibition of cyclin E-cdk2 by intact p21 and by p21 without the cyclin-
binding site.

Fig. 1 indicates that with 2 |ig of GST-CDC25A the kinase activity is linear beyond
10 minutes. Thus for the inhibition assays the kinase reaction was stopped at 10 minutes.

Fig. 2A shows that compared to wild type p21, p21A17-24 (deletion in one of the
cyclin binding motif, Cyl) is almost 50 fold weaker in its inhibitory potency, while
p21A53-58 (deletion in the cdk binding K motif) is about 8 fold weaker. This surprising
result indicates that for certain substrates the Cyl motif of p21 is even more important than
the K or Cy2 motifs for inhibition of cyclin E-cdk2. In the future, we wiU examine
whether the same holds for cyclin A-cdk2 (which might prefer the Cy2 motif near the C
terminus of p21 over the Cyl motif in the N terminal half). We will also examine the
inhibitory reaction with a mutant substrate lacking a Cy motif, GST-CDC25A2 (18).

5

Collectively these experiments will indicate the relative importance of each Cy mo^s (both
on the inhibitor and on the substrate) for kinase inhibition. In addition these experiments
will reveal whether there is specificity encoded in the Cy motifs (Cyl or Cy2) with regard
to which cyclin (A or E) is preferentially targeted.

Fig. 2B shows that PSKX), the 12 amino acid peptide containing the Cy motif
(ACEELEGPVDSE), inhibits the activity of cyclin E-cdk2 with an IC50 of about 200

ng/25 jxL (6 |xM). In contrast the mutant peptide, PS 101 (ACRRLKKPVDSE) is inactive

up to 10000 ng (300 ^M). In comparison an IC50 of 200 nM for PSIOO was reported
when cyclin E/cdk2 was used to phosphorylate GST-RbC (the C terminal portion of the
retinoblastoma protein) (5). The 12 fold decrease in potency of PSIOO on CDC25A could
be accounted by the Cy motif on CDC25 A (the substrate used here) having a higher affinity
for cyclin E/cdk2 than the Cy motif on Rb. This question will be addressed by using
peptides that encode Cy motifs from other substrates of cdks (Rb, CDC25A and
CDC6/Cdcl8).

Mutagenesis of the Cy motif of p21.

Of the three methods considered, we had initially focused on a PCR based strategy
to change the first R (R1 to A) and the second R (R2 to A) of the Cy motif of GST-p21N.
However, we failed to obtain the correct mutant by this method.

We then tried the cassette based mutagenesis strategy. This is the method that we
hope to optimize because if successful, cassettes with degenerate oUgonucleotides will
provide an easy way to produce a library of mutants in die Cy motif of GST-p21N. Two
types of ligations have been tried using the R1 to A and R2 to A mutagenic oligos for
optimization. Unphosphorylated oligos have been ligated to un-phosphatased GEX-p21N
plasmid cut open with Hindlll and Blpl. For comparison, kinases oligo-cassette has been
ligated to GEXp21N plasmid linearized by HindHI and Blpl and de-phosphorylated with
shrimp alkaline phosphatase. Using the first ligation approach we obtained mutants at an
unusually low frequency (2 out of 1 8), which is sub-optimal for making a library of
mutants. Currently we are awaiting the results from the second ligation approach.

Determination of the part of PCNA that interacts with p21 and Fenl.

In vitro transcribed -translated PCNA (wild type and mutants) were bound to
glutathione agarose beads coated with GST-p21 , GST-FenlC and (3ST (negative
control). No difference was seen in the binding of the mutants to p21 or Fenl (not
shown), a result that contradicts with the published report (17). We reasoned that non¬
radioactive wild-type PCNA subunits in the rabbit reticulocyte lysate may form heteromers
with the radiolabeled mutants and facilitate their association with p21 or Fenl giving a false
positive result. Wild type and mutant PCNA were therefore synthesized in E. coli (which
do not contain wild type eukaryotic PCNA), lysates containing the PCNA prepared, and
the binding of PCNA to GST, GST-p21 and GST-FenlC assayed. Fig. 3 shows that wild
type PCNA and several of the mutants successfully bound to p21 and to Fenl. However,
in agreement with published results, a mutation that changed the QLGI in the inter-domain
loop of PCNA to AAAA, resulted in PCNA that failed to bind to p21 and Fenl.

Therefore we have at least one derivative of PCNA that has lost interaction with
Fenl and p21. PCNA with QLGI changed to AAAA has been purified to homogeneity
over nickel-resin and Mono-Q columns (Fig. 4) preparatory to analyzing its effect on
polymerase delta and its effect on genomic stability of direct repeats in an in vitro DNA
replication reaction. In addition, finer mutations will now be made in and around the QLGI

6

region of PCNA to produce mutant forms of PCNA that selectively lose interaction with
p21, Fenl or polymerase delta. If such mutant forms of PCNA ^e found, over-expression
of such proteins during in vitro or in vivo replication reactions will address the relative
roles of these interactions in (a) maintenance of genomic stability and (b) the effect of p21
on DNA replication and genomic stability.

CONCLUSIONS

The cyclin-binding motif of p21 appears to be much more important than the cdk
binding K motif for inhibiting the phosphorylation of substrates like CDC25A that contain
exactly similar cyclin-binding Cy motifs. Future experiments will establish whether Ais is
true by quantitative comparisons between substrates with different Cy motifs and inhibitors
(p21 or synthetic peptides) with different Cy sequences. Preliminary experiments are still
in progress to help select the optimal method of mutagenesis to make a library of mutant Cy
motif in p21. The best method we have till now is successful at the rate of 2 out of 18
clones. If further improvement is not obtained by the other methods, we shall use this
method to generate the mutants and test them. Mutant forms of PCNA have been generated
that fail to interact with both p21 and Fenl. Finer mutations in PCNA over this region will
allow us to distinguish the relative importance of PCNA-pol delta, PCNA-Fenl and
PCNA-p21 interactions for the inhibition of DNA replication and the maintenance of
genomic stability

FIGURE LEGENDS

Fig. 1 Rate of phosphorylation of a substrate GST-CDC25A by cyclin E-cdk2 under
conditions described in the text. Y-axis: cpm of 32P incorporated in the GST-CDC25A
band. X-axis: Time of kinase reaction.

Fig. 2 Inhibition of cyclin E-cdk2. (A) Addition of indicated amounts of GST-p21
(squares), GST-p21 A17-24 (circles; deletion of cyclin E binding motif) and GST-p21

A53-58 (diamonds; deletion of cdk2 binding motif). The amount of 32P incorporated into
GST-CDC25A at 10 minutes (initial velocity conditions) is expressed as percentage of 32P
incorporated in the uninhibited reaction (100% kinase activity). (B) Addition of synthetic
peptides PSIOO (squares; wild type Cyl motif) and PSIOI (diamonds; mutant Cy motif).
The rest is the same as in part A.

Fig. 3 QLGI PCNA fails to bind to Fenl or to p21. PCNA bound to GST (GEX),
GST-FENIC (GEX-FENIC) and GST-p21 (GEX-p21) was visualized by immunoblotting
with anti-PCNA antibody (top). 10% of the PCNA input into the reactions is directly
visualized by immunoblotting (bottom), wt: wild-type PCNA. VDK: Vall88, Aspl89
and Lysl90 of PCNA changed to Ala. QLGI: Glnl25, Leul26, Glyl27 and Ilel28 of
PCNA changed to Ala. SHV: Ser43, His44 and Val45 of PCNA changed to Ala. LAPK:
Leu251, Pro253 and Lys254 of PCNA changed to Ala.

Fig. 4 QLGI 125 to AAAA form of PCNA purified to homogeneity and visualized by
Coomasie Blue staining after SDS-polyacrylamide gel electrophoresis.

7

GEX

D I GEX-FEN1C
^ I GEX-P21

LU

CD

lil^

>S: ik

UJ UJ
CD CD

O

T“

Z T-
UJ CM

u. a
X >!: X

UJ UJ UJ

CD CD CD

QLGI

Wt

SHV

LAPK

VDK QLGI Wt SHV LAPK

0.1 X Input

Fig. 3

11

Purification of PCNA Mutant

QLGi

Fig. 4

QLGI

12

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complex Nature 382, 325-331

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9. Waga, S. and Stillman, B. (1994). Anatomy of a DNA replication fork revealed by
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13. Vallen, E. A. and Cross, F. R. (1995). Mutations in rad27 define a potential link between g(l)
cyclins and dna replication Molecular & Cellular Biology 15, 4291-4302

14. Reagan, M. S., Pittenger, C., Siede, W., Friedberg, E. C. (1995). Characterization of a
mutant strain of saccharomyces cerevisiae with a deletion of the rad27 gene, a structural
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rthl 5' to 3' exonuclease for the stability of simple repetitive dna Science 269, 238-240

16. Chen, J., Chen, S., P., S., Dutta, A. (1996). p21 disrupts the recruitment of Fenl to the
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